The proposal is focused on the mechanisms by which scavenger receptor BI (SR-BI) stimulates the flux of free cholesterol (FC) between cells and lipoproteins. FC removal from peripheral cells and FC and cholesteryl ester (CE) uptake by liver cells are the first and last steps, respectively, in the process of reverse cholesterol transport (RCT). SR-BI has been shown to participate in both steps and plays an important role in protecting against atherosclerosis development in mouse models. This application has 3 Specific Aims to evaluate the mechanisms and physiological significance of SR-BI-mediated cholesterol flux. Aim 1 has 4 goals that will test SR-BI activities in cell culture and in vivo in mouse models. Goal 1 will use mutagenesis and functional studies to extend our understanding of recently identified SR-BI separation of function (SoF) mutations that have lost some but not other activities of SR-BI. Goal 2 will test wild type and SoF mutants for the ability to stimulate FC clearance in an in vivo foam cell model. Goal 3 will test SR-BI and SoF mutants for the ability to promote HDL FC and CE uptake, CE hydrolysis, and transport of FC to bile using a mouse liver perfusion system. Goal 4 will test SR-BI/CD36 chimeras and SR-BI SoF mutants for the ability to restore normal adrenal plasma membrane function and structure in SR-BI-deficient mice using adrenal-specific transgene expression. Aim 2 has 3 goals that test the activity of SR-BI on membrane organization and test the effect of membrane organization on SR-BI activity. Goal 1 will use electron microscopic approaches to identify SR-BI domains and activities that are necessary for receptor clustering on microvillar extensions of the plasma membrane. Goal 2 will test the hypothesis that fluid membrane domains promote SR-BI-mediated FC flux. These experiments will incorporate well-characterized sterol analogues into cells to ask whether SR-BI-mediated FC flux is enhanced by increasing the fraction of the plasma membrane in the fluid versus liquid-ordered state. Goal 3 will employ fluorescence microscopy with membrane domain probes in living cells to test the hypothesis that SR-BI modulates membrane domain organization via uptake of HDL cholesterol. Aim 3 will directly test the effect of SR-BI on FC flux and the organization of FC in model membranes in which SR-BI is incorporated into reconstituted multilamellar and large unilamellar vesicles. These studies will provide new and important information about SR-BI-mediated cholesterol flux and its role in RCT.